This invention relates to gold sol coated with alkanethiols and alkanethiol derivatives to provide groups on the sol available for the binding or linking of binding moieties such as antibodies, antigens, or ligands to the gold sol. In addition, the use of di- and tri-thiol compounds bound to gold sol facilitate the passive adsorption of these binding moieties. This invention also relates to gold sol coated with thiolated binding moieties, including antigens, antibodies, or carrier molecules, which can be attached to relevant ligands. Also included is the process for coating the gold sols with such thiol compounds, the use of coated sols in immunological and immunocytological diagnostic tests and test kits incorporating such coated gold sols.
Test methods for the diagnosis of various diseases are constantly being improved. Currently, immunological methods are among the most sensitive methods used to detect the presence of antigens or antibodies in samples. These assays are well known to those skilled in the art of immunodiagnostics. Generally, an immunological assay consists of an assay wherein a monoclonal or polyclonal antibody is used to capture an antigen in the sample and a second antibody containing a label, such as a fluorescent compound or an enzyme, immunochemically reacts with the antigen-antibody complex. The resulting labelled antibody-antigen-antibody complex is detected. Variations on this basic assay are common, such as the use of only one reactive antibody in the test, the competitive inhibition method, or the use of particles as labels that allow an agglutination reaction to be read. Another variant is the use of microparticles coated with either an antigen or an antibody, which after formation of a complex with the analyte and a second appropriately labeled binding partner, gives a positive reaction.
Gold sol microparticles are used in an assay method known as a sol particle immunoassay (SPIA). In this assay, a solution containing the gold sol coated with an appropriate binding partner, either an antibody or an antigen, is reacted with a sample to bind to its binding partner. In this process, complexes are formed that can be detected, usually due to their change in color.
Uncoated gold sol particles, and other colloids, will undergo agglomeration when exposed to low concentrations of salt, and quickly precipitate out of solution. Therefore, the coating of gold sol with an appropriate binding partner serves two functions. The first is to provide appropriate immunological binding activity and the second is to protect against agglomeration, which would of necessity occur in buffers designed to optimize for immunological reactions. Since not all proteins or polymers will protect the gold sol completely from salt-induced agglomeration, an overcoating step is usually performed with a protein or polymer that is known to be capable of completely protecting the gold sol from salt-induced agglomeration. Such an overcoating step is a well known practice in adsorbing antibodies and antigens to plastic substrates and has been shown to increase the stability of the coated material.
The overcoating also serves to reduce nonspecific interaction of the gold sol with sample components. This nonspecific interaction is a significant problem when using antibody coated latex particles in diagnostic assays, and is probably a manifestation of the nonspecific serum interference observed in many, if not all, immunoassays, regardless of format. In some cases it is permissible to change the overcoating protein or polymer to minimize interference in specific systems. Additives to the sol medium such as guanidine hydrochloride or urea are useful. Occasionally, "non-specific" interference can be pinpointed to serum heterophile activity and is eliminated by the addition of whole animal serum.
A serious drawback in the passive adsorption of the desired binding partner to gold colloid has been that such direct coating is often unsuccessful. The physico-chemical mechanisms of passive polymer adsorption to colloids in general is a poorly understood process. Passive adsorption of antibodies to gold sol may result in a coated sol which is poorly protected from salt-induced agglomeration and which cannot be further protected by overcoating. It may also result in a coated sol with poor immunological activity, presumably due to the incorrect orientation of adsorbed antibody, or it may result in the antibody-induced agglomeration of the sol itself. Finally, the binding partner of choice may simply not bind to the gold sol. The net result of these problems is that few biological reagents useful in other diagnostic formats can be used in the production of gold colloid reagents of diagnostic quality.
What is needed in the art is a method for covalently attaching binding partners, proteins, carbohydrates or ligands, to the gold sol so that the uncertainties of the passive adsorption characteristics or the necessity of making binding partner and "good coating" carrier conjugates for passive adsorption may be eliminated. In particular, such a sol would be significantly more useful if it were refractive to salt-induced agglomeration even in the absence of a coated binding partner. The ability to change the physico-chemical surface properties of the sol would, at the same time, make it possible to minimize the sometimes undefined sample-sol interactions responsible for non-specific interference in immunochemical diagnostic assays and background problems in immunocytochemical assays.
What is also needed is the ability to facilitate the passive adsorption of biological polymers to gold sols. Thiolation of antibodies and polymers can increase their ability to bind to gold sols as evidenced by increased resistance to salt-induced agglomeration. Such a capability may prove useful for those antibodies which bind well to gold sols, but lose significant amounts of their activity in doing so as well as for antibodies which simply do not bind the gold sol in an underivatized state. An alternative method of changing the physico-chemical characteristics of the sol surface in order to facilitate antibody binding is the coating of the sol with di-thiol or tri-thiol compounds. Such an intermediate coating can significantly change the passive adsorption properties of antibodies to the coated sol.